Acetoxymethyl esters

Rabbit peritoneal neutrophils were harvested and their release of p-glucuronidase was measured at 37°C, as described previously (13). For indo-1, neutrophils were washed twice in a calcium-free buffer, then loaded with 15 indo-1 acetoxymethyl ester (24) for 40 min at 37 C at a density of 5 x 10 cells/ml. The cells were then washed twice more in calcium-free buffer, resuspended to a density of 1 X 10 cells/mL, and kept on ice. Prior to fluorometry, cells were diluted 4x with the appropriate buffer at 37 C. For CTC, neutrophils were incubated with 20 pH CTC at 37°C in the spectrofluorometer cuvette. All measurements were carried out using an SLH-Aminco SPF 500C fluorospectrometer interfaced with an IBM PC microcomputer. 

FIG. 3. Confocal images showing the location of the SR in live myocytes within an intact, small diameter (< 250 nm passive diameter), pressurized (70 mmHg) artery from the rat mesenteric artery arcade. The artery was loaded with Fluo-4 as the membrane-permeant acetoxymethyl ester. Some of this high-affinity, Ca2+ indicator dye is often sequestered in the SR (cf. Goldman et al 1990). The SR can then be readily visualized, especially when [Ca2+]CYx is low (as in the panels at 0 and 6.8 s), because the intra-SR dye is saturated with Ca2+, and fluoresces brightly. This artery was treated with 1.0 fim phenylephrine (PE), which caused the [Ca2+]CYT level to oscillate asynchronously in the cells seen in the centre of the panel. The cell outlines are clearly visible when [Ca2+]CYT tiscs, as in the panels at 3.4 and 10.2 s. Note that nearly all of the SR (the very bright areas, especially in the 0 and 3.4 s panels) lies parallel to, and immediately beneath the PL (from Miriel at al 1999, with permission). 

ADCC ADP AM ara-C ATP BAPTA antibody-dependent cellular cytotoxicity adenosine diphosphate acetoxymethyl ester cytosine arabinoside adenosine triphosphate bis- (o-aminophenoxy)-ethane-N,N,N, N -tetraacetic acid... 

By keeping the same fluorophores as in 17 and 18a, but reducing the cavity size of the ionophore, one obtains Mag-Indol (19) and Mag-Fura2 (20) (Figure 2.9) which are selective for magnesium.(48) All the chelators 17 to 20 are commercially available under the nonfluorescent acetoxymethyl ester form so that they are cell permeant and they recover their fluorescence on hydrolysis by enzymes.<48)... 

The acetoxymethyl ester of benzylpenicillin is penamecillin (Havapen, 1). It is an almost white, free-flowing powder lacking the characteristic odour and taste which make penicillins generally unpalatable when administered in tablet or... 

A new class of lipophilic ferrichrome analogs carrying acetoxymethyl ester moieties 203 has been synthesized and shown to penetrate rapidly through cell membranes. Intracellular esterase mediated hydrolysis transformed the lipophilic termini from hydrophobic to hydrophilic. The intracellular retention was visualized in hepatoma cells by labeling these analogs with a fluorescent naphthy-imide probe . Their fluorescence analogs retained their fluorescent properties for extended periods in comparison to hydrophobic derivatives 204 lacking the cleavable substituents. 

A product that is widely used, and one that has also grown in importance since the advent of high throughput screening methods, is the AM (acetoxymethyl) ester of the fluoran derivative, Fluo-3 (3.67). This product, Fluo-3AM, does not itself bind Ca but is readily hydrolysed to Fluo-3 by endogenous esterases once the dye is in the cells. 

Fluorescent dyes such as calcein-AM and rhodamine derivatives have been demonstrated to be P-gp substrates (400-407). These compounds can be used in any competition assay in which the test compound is added with these dyes. Any reduction in the dye efflux would be indicative of the inhibitory properties of the test compounds toward P-gp. Both rhodamine 123 and calcein-AM have been used in high-throughput assays, including the NCI assay, to screen large numbers of compounds as inhibitors of P-gp in several cell types. Calcein-AM itself is a weakly fluorescent molecule. When the acetoxymethyl ester group is cleaved by... 

The acetoxymethyl ester form of the calcein dye (calcein-AM) has been widely used as an assay to measure viable cells for microscopy, flow cytometry, and some plate-based methods. Calcein-AM is a cell-permeable non-fluorescent compound added to cells as a solution in DMSO. Non-specific esterase activity present in viable cells removes the lipophilic AM group, resulting in conversion of the non-fluorescent calcein-AM into a green fluorescent calcein molecule that is generally retained inside viable cells. 

In addition, the Botana group observed that gambierol also stimulated a cytosolic calcium increment by using the dye Fura-2 acetoxymethyl ester (Fma-2, AM) (Grynkiewicz, Poenie, and Tsien 1985). Fig. 1.8 shows that gambierol induced no calcium increment when this cation was omitted from the incubation medium. This result indicates that extracellular calcium was the source of the gambierol-evoked cytosolic calcium increase (Louzao et al. 2006). 

BCECF-acetoxymethyl ester (Molecular Probes, Eugene, OR) is used at 40 ig/ml final concentration in culture medium, for 15 min at 37°C. After washing, tumor cells are resuspended in HEPES-buffered serum free medium without phenol red (assay medium)... 

More recently reported active esters are the different acyloxymethyl esters [129]. The pharmacology of penicillin G acetoxymethyl ester (Penamecillin) was investigated in 1966 [130]. A further derivative is the acetoxymethyl ester, or preferably the pivaloyloxymethyl ester, of am-... 

Esterase substrates have long been used as a measure of cell membrane integrity, with the fluorescent product being the marker retained. The one that has been used most often with fish cell lines is 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM)174 CFDA-AM diffuses into cells rapidly and is converted by... 

After oral administration to the dog, penamecillin (VII), which is the acetoxymethyl ester of benzylpencillin, produces prolonged benzylpencillin serum levels.According to pharmacokinetic evidence O, the penamecillin is slowly absorbed unchanged throughout the intestinal tract and then is rapidly hydrolyzed to benzylpenicillin by nonspecific esterases. Compound VIII is an example of the hitherto unknown penicillin aldehydes for which a general synthesis was developed. 

Penem[2- C] FCE 22101, 219, a j -lactam antibiotic, has been synthesized in eight steps in overall radiochemical yield of 21%, 98% radiochemical purity and specific activity of 641 MBqmmoP (17.3 mCi mmol ) using sodium salt of [1- C]glycolic acid as the starting labelled material (equation 91). The acetoxymethyl ester FCE 22891 220 has been prepared in 41% yield by condensing 219 with bromomethyl acetate. 

Pcnamecillilt. 3,3-Di methyl-7-oxo-6-[(phenylace-tyl)amino]-4-thla-l-azabicyclo[3.2.0]heptane-2-ca rboxy lie acid (acetyloxyfmeihyl ester penicillin G hydroxymethyl ester acetate acetoxymethyl benzylpenicillinate benzylpenicillin acetoxymethyl ester Wy-20788 Havapen. C.,HKN,06S mol wt 406.48. C 56.14%. H 5.46%, N 6.89%, 6 23.62%, S 7.89%. Semi-synthetic antibiotic related to penicillin. Prepn Jansen, Russell. J. Chem. Soc. 1965, 2127 eidem, Brit. pat. 1,083,479 corresp to U,S. pet. 3,250,679 (1965, 1966 to John Wyeth A Brother). 

In addition, they used another fluorescent dye, Fura-2 acetoxymethyl ester (Fura-2, AM) in order to test cytosolic calcium changes induced by gambierol. They detect a calcium increase depending on extracellular sodium and abolished in a Ca -free solution (Figure 28.15). Then they reach to the conclusion that gambierol induced a sodium-dependent calcium entry. Inhibitors such as dichlo-... 

FIGURE 31.4 Intracellular acidification caused by palytoxin in primary cultures of cerebellar granule cells as assessed with the pH-sensitive fluorescent indicator BCECF-AM (2 7 -bis(carboxyethyl)-5(6)-carboxyfluo-rescein acetoxymethyl ester). (Modified from Vale, C and Ares, I.R., in Phycotoxins Chemistry and Biochemistry, Botana, L. (Ed.), Blackwell Publishing, 2007, pp. 95-118.)... 

The acetoxymethyl ester of calcein (Calcein-AM) is a highly lipid soluble and nonfluorescent compound that can rapidly penetrate cell plasma membranes. Once Calcein-AM is taken up into the cells, the ester bond can be quickly and irreversibly converted to hydrophilic, nonpermeable and intensively fluorescent free acid form, Calcein. When traversing the cell plasma membrane, Calcein-AM can be pumped out of the cells by P-gp. Inhibition of P-gp causes an increase in the cellular level of Calcein-AM and subsequently the appearance of fluorescent Calcein. Calcein AM uptake studies in P-gp expressing cells have been extensively used as a screening tool for P-gp inhibitors (Polli et ah, 2001). 

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